The present invention relates generally to a method for the generation of a composition of matter comprised of a unique profile of primary aliphatic alcohols, having 24 to 30 carbon atoms, from a novel natural sourcexe2x80x94a wax secreted by the Chinese wax soft scale insect, Ericerus pela, which belongs to the family Coccidae. The present invention includes the composition of matter, referred to herein as xe2x80x9cpolycosanol(s)xe2x80x9d produced by the method of the invention. The polycosanol composition produced according to the method of this invention is comprised of primarily four primary aliphatic alcohols, tetracosanol, hexacosanol, octacosanol and triacontanol. The invention also includes the use of this composition of matter for the prevention and treatment of obesity, syndrome X, diabetes, hypercholesterolemia, atherosclerotic complications, ischemia and thrombosis.
Polycosanols are a class of primary aliphatic alcohols having 20 to 40 (C20-C40) carbon atoms. They are widely distributed in germs, kernels and other components of nuts, seeds, fruits and cereals (Kawanishi et al. (1991) J. Amer. Oil Chemist Soc. 68:869-872), in Greek olive oils (Dimitrios et al. (1983) Grasas Aceites 34:402-404) and in apple wax (Belding et al. (1993) Hertscience 28:90). Polycosanols are also present, in very small amounts (less than 0.1%), in wheat grain, in the form of the long chain alkyl esters of fatty acids. The major compounds present in wheat grain include, palmitoyl hexacosanol and arachidoyl, palmitoyl and behenoyl tetracosanol (Ohnishi et al. (1986) Cereal Chem 63:193-196).
Polycosanols, both as the free alcohols and the esters of fatty acids, have been isolated from many different genera and species of plants, including from the species Achillea biebersteinii (Oskay and Yeslada (1984) J. Nat. Prod. 47:742), Calamagrostis arundinacea (Solberg (1976) Acta Chem. Scand Ser. B. Org. Chem. Bioche. 30:786-787), Emilia sonchifolia (Srinivasan and Subramanian, (1980) Fitoterapia 51:241-244), Heliotropium digynum (Ismail et al. (1984) Fitoterapia 55:110-112), Hypericum perforatum (Brondze (1983) J. Nat. Prod. 46:940-941), Tragopogon orientalis (Krzaczek et al. (1988) Acta Soc. Bot. Pol. 57:85-92), and from the genera of Triceae (Tulloch (1981) Can. J. Bot. 58:2602-2615). Polycosanols have also been isolated from various parts of many different genera and species of plants, including from the bark of various Acacia species (Banerji and Nigram (1980) J. Indian Chem. Soc. 57:1043-1044); from the stem of Anisomeles indica (Dobhal et al. (1988) Fitoterapia 59:155); from the leaves of Cordia rothii (Behari et al. (1980) Acta Cienc. Indica Chem. 6:226-228), Hibiscus cannabinus (Makhsudova (1979) Chem. Nat. Compd. 15:186) and Holigarna arnottiana (Prakash and Banerji (1979) Fitoterapia 50:265-266); from the roots of Talinum paniculatum (Komatsu et al. (1982) Yakugaku Zasshi 102:499-502); from the leaves and roots of Gymnosporia Montana (Kumar and Srimannarayana (1981) J. Nat. Prod. 44:625-628); from the aerial parts of Cymbopogon citrates (Olaniyi et al. (1975) Planta Medica 28:186-189), E. merifolia (Baslas and Agarwal (1980) Indian J. Pharm. Sci. 42:66-67) and E. Peplus (Rizk et al. (1980) Fitoterapia 51:223-228), Portulaca suffruticosa L. (Joshi et al. (1987) Herba Pol. 33:71-74) and Youngia denticulate (Arai et al. (1982) J. Pharm. Soc. Jp. 102:1089-1091); and from the heartwood of Melia birmanica (Banerji and Nigam, (1981) Fitoterapia 52:3-4). Polycosanols have also been isolated from carnauba wax from exudates of the leaves of the palm tree Copernicia cerifera (Pinto and Bento (1986) Rev Soc Bras Med Trop 19:243-5), from the leaf wax of Eulphorbia helioscopia (Nazir et al. (1993) Xeischrift fur Naturforschung. Section C Biosciences 48:5-9), from epicuticular waxes of genera of Gramineae (Tulloch (1981) Can. J. Bot. 59:1213-1221), and from and the latex of Euphorbia pseusocactus (Awad et al. (1993) Fitoterapia 64:553) and Euphorbia thymifolia (Agarwal and Maslas (1981) Indian J. Pharma. Sci. 43:182-183). Finally, polycosanols have been isolated from the Korean indigenous plant Echinosophora koreensis (Kang and Kim (1987) Arch Pharmacal Res. 10:67-68).
Bertholet has described a method for preparing polycosanol compositions by means of the saponification of plant wax from rice bran wax, carnauba wax and jojoba oil. (Bertholet, U.S. Pat. No. 5,159,124 (1991)). In the method described by Bertholet, the plant wax was first dissolved in an organic water immiscible solvent, such as butanol or pentanol, and then hydrolyzed using an aqueous solution of an alkaline earth metal hydroxide. The fatty acid by products of the saponification reaction are soluble in the alkaline aqueous layer and the polycosanol alcohol product remains in the organic layer, which contains  less than 10% fatty acids and  greater than 90% alcohols. The overall yield of the reaction was approximately 50%. The composition of the polycosanol product is dependent on the origin of the plant wax.
N-hexacosanol has been isolated from wool wax hydrosylate mixtures using gel permeation chromatography (Steel et al. (1999) International Publication No. WO 99/48853).
Polycosanol compositions isolated from rice bran wax have been formulated with phytosterol from vegetable oil and used for reducing cholesterol levels. The aliphatic alcohol profile of this material is approximately 23-33% total polycosanol. Triacontanol is the major compound (8-9%), followed by octacosanol (5-6%) and tetracontanol, hexacosanol, dotriacontanol and tetratriacosanol (2-5% each). (Sorkin Jr. (1999) U.S. Pat. No. 6,197,832, and Sorkin Jr. (1998) U.S. Pat. No. 5,952,393). Octacosanol isolated from Sinach has been formulated with other ingredients as a nutritional powder for boosting energy. (Gaynor U.S. Pat. No. 5,744,187 (1996)).
Sugar cane provides a major natural source of commercial polycosanol products (Ali et al. (1979) Egypt J. Pharm. Sci. 18:93-99). The long chain aliphatic alcohols are located primarily in the wax layer of sugar cane, with octacosanol being the predominant compound (Nagata et al. (1994) Breeding Sci. 44:427-429) (See Table 1, below). Aliphatic alcohols from sugar cane wax can be extracted directly with a supercritical fluid, an organic solvent or an alcohol to obtain a mixture with octacosanol (7-10%) and triacontanol (0.4-1%) as the major components (Inada et al. (1986) U.S. Pat. No. 4,714,791). A mixture of higher primary aliphatic alcohols, having from 24 to 34 carbon atoms has been obtained by saponification of sugar cane wax. (Laguna et al. (1996) U.S. Pat. No. 5,856,316). The saponification reaction described included melting the sugar cane wax, forming a homogeneous phase with an alkaline earth hydroxide (5-30%), extracting with an organic solvent and recrystallizing from an organic solvent. The profile of the material included octacosanol as the major component (60-70% content), followed by triacontanol (10-15%), hexacosanol (5.5-8.5%), dotriacontanol (4-6%), heptacosanol (2-3.5%), tetratriacontanol (0.4-2.0%), nonacontanol (0.4-1.2%) and tetracosanol (0.5-1.0%). This material has been formulated with acetylsalicylic acid and used for the treatment of hypercholesterolemia, atherosclerotic complications, gastric ulcers and to improve male sexual activity. (Laguna et al. (1996) U.S. Pat. No. 5,856,316).
Ericerus pela, which belongs to the family Coccidae (Ben-Dov and Hodgson, (1997) Soft scale insects; their biology, natural enemies and control. Vol. 7A. Elsevier Science Publishers, Amsterdam), is an insect indigenous to southern China, having the common name white wax scale. (Zhang (1987) Scientia Silvae Sinica 23:383-385, Cen and Ji (1988) Insect Knowledge 25:230-232). This insect has a high economic value in China (Chen (1999) World Forest Res. 12:46-52), due to its ability to produce wax and its high nutritional value (Zhao et al. (2001) Entomological Knowledge 38:216-218). The female lays over 7000 eggs on average (Park et al. (1998) Korea J. Applied Entomology 37:137-142) and egg hatching is directly related to wax production (Chen et al. (1997) Forest Res. 10:149-153). The reproductive capability of Ericerus pela can be impacted by sex ratio, lifespan, habitat and other ecological conditions. (Zhang et al. (1993) Entomological Knowledge 30:297-299). The eggs from this insect contain a high percentage of proteins (40-55%) and amino acids (30-50%) and are nutritious and safe for human consumption. (Ye et al. (2001) Forest Res. 14:322-327). The insect can be raised on 200 different species of host plants belonging to 98 genera and 36 families. (Chen and Li (2001) Forest Res. Beijing 14:100-105). The host plants provide not only their habitat and reproductive sites, but also serve as their food source. (Chen et al. (1997) Forest Res. 10:415-419). The average amount of wax production is affected by the host plant species (Chen et al. Forest Res. 11:285-288), geographic varieties of the insect (Chen et al. (1998) Forest Res. 11:34-38) and climate conditions, particularly temperature, dryness and intense sunshine. (Liu et al. (1998) Forest Res. 11:508-512). Ericerus pela has been produced in commercial forest plantations and the conditions and value of crop production have been reported. (Liu et al. (1996) Forest Res. 9:296-299).
The wax from Ericerus pela is secreted from the wax gland of both male and female insects. (Tan and Zhong (1992) Zoological Res. 13:217-222). The composition of the insect wax has been analyzed by GC/MS and determined to be hexacosyl hexacosanoate (55.16%), hexacosyl tetracosanoate (22.36%) and hexacosyl octacosanol (16.65%). (Takahashi and Nomura (1982) Entomol. Gen. 7:313-316). The wax has traditionally been used for bleeding, pain relief, wound healing, coughing and diarrhea. (Li (1985) World Animal Review 55:26-33). Saturated long chain fatty alcohols have also been found in other insects, including Drosicha corpulenta (Hashimoto and Kitaoka (1983) Appl. Entomol Zool. 17:453-459).
Bee wax also contains a significant quantity of long chain primary alcohols in both the free and esterified forms. Polycosanol compositions isolated from bee wax contain 24 to 34 carbon atoms (C24-C34) comprised of tetracosanol (9-15%), hexacosanol (12-18%), octacosanol (13-20%), triacontanol (20-30%) and dotriacontanol (13-21%). (Hernandez et al., U.S. Pat. No. 6,235,795 (1994)). Bee wax, formulated with olive oil, xcex2-sitosterol and an extract from Coptis chinensis, has been used for the treatment of diaper rash (Niazi, U.S. Pat. No. 6,419,963 (2001)) and as a pharmaceutical and cosmetic carrier (Xu, U.S. Pat. No. 5,817,322 (1996)). Polycosanols isolated from bee wax also show anti-ulcer and anti-inflammatory activity. (Mas (2001) Drugs of the Future, 26:731-744; Carbajal et al. (1996) J. Pharmacy and Pharmacol. 48:858-860; Hernandez et al., U.S. Pat. No. 6,235,795 (1994)).
Polycosanol compositions isolated from bee wax upon saponification contain primarily octacosanol (13.0-20.0%), triacontanol (20-30%), dotriacontanol (13-21%), hexacosanol (12-18%), tetracosanol (9-15%) and tetratriacontanol (1.5-3.5%). (Hernandez et al., U.S. Pat. No. 6,465,526 (2000)). In the method reported by Hernandez et al., the saponification reaction was performed in the homogeneous phase using a 4-7:1 wax:base ratio. After hydrolysis, the polycosanols were extracted with organic solvents to produce a product that contained 80-98% total polycosanols in a yield of approximately 30% from bee wax. (Hernandez et al., (1994) U.S. Pat. No. 6,235,795). As noted above, this material showed both anti-ulcer and anti-inflammatory activity. Polycosanol compositions obtained from the saponification of bee wax have also been formulated with acetyl salicylic acid for use in the treatment of hypercholesterolemia, atherosclerotic complications, gastric ulcers and to improve male sexual activity (Granja et al., U.S. Pat. No. 5,663,156 (1994)).
The polycosanols in bee wax have also been extracted directly with organic solvent without saponification. (Perez, U.S. Pat. No. 6,225,354 (1999)). This material contained octacosanol (30-60%), triacosanol (16-26%), dotriacontanol (13-22%) and hexacosanol (7-12%) as the major components and has been shown to be effective in the treatment and prevention of hypercholesterolemia related diseases. (Perez, U.S. Pat. No. 6,225,354 (1999)).
Polycosanol compositions isolated from sugar cane have been shown to lower cholesterol levels in both animal and human models. (Menedez et al. (2000) Br. J. Clin. Pharmacol. 50:255-262; Arruzazabala et al. Braz. J. Med. Biol. Res. 33:835-840; Crespo et al. (1999) Int. J. Clin. Pharmacol. 19:117-127; Gouni-Berthold and Berthold (2002) Am. Heart J. 143:356-365; Alcocer et al. (1999) Int. J. Tissue React 21:85-92). Modulation of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase was observed in a celline model, but not in a pure enzyme inhibition assay. (Menendez et al. (2001) Arch. Med. Res. 32:8-12). Instead of inhibiting of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase, as most cholesterol lowering drugs, polycosanol may have different mechanism of action, such as the down regulation of HMG-CoA reductase production in gene expression and/or at the proteomic level. (McCarty (2002) Med. Hypotheses 59:268).
Older patients with hypertension and Type II hypercholesterolemia, treated with polycosanol compositions isolated from sugar cane at a dosage of 20 mg/day for twelve months, showed significantly decreased TC, LDL, LDL/HDL and TC/HDL levels, and increases HDL levels. (Castano et al. (2002) Drug R D. 3:159-172; Castano et al. (2001) Int. J. Clin. Pharmacol. 21:43-57). Even at a dosage of 5-10 mg, polycosanol compositions isolated from sugar cane showed a significant benefit in hypercholesterolemia postmenopausal women (Mirkin et al. (2001) Int. J. Clin. Pharmacol. 21:31-41; Castano et al. (2000) Gynecol. Endocrinol. 14:187-195) and in high coronary risk older patients (Castano et al. (2001) J. Geontol A Biol. Sci. Med. Sci. 56:M186-192; Castano et al. (1999) Int. J. Clin. Pharmacol. 19:105-116). In summary, it has been proposed that polycosanol compositions isolated from sugar cane could potentially provide a new treatment for cardiovascular disease with equal or better clinical output than simvastatin, pravastatin, lovastatin, probucol and acipimox. (Janikula (2002) Altern. Med. Rev. 7:203-217).
Polycosanol compositions have also been shown to exhibit anti-thrombic effects (Carbajal et aL (1998) Pharmacol. Res. 38:89-91), with significant inhibition of platelet aggregation (Arruzazabala et al. (1993) Thromb Res. 69:321). Additionally, unlike aspirin polycosanol did not affect the platelet anti-aggregating enzyme PGI2, but rather inhibited platelet aggregating enzyme thromboxane B2 (TXB2). (Carbajal et al. (1998) Prostaglangins Leukot. Essent Fatty Acids 58:61-64). This makes a combination therapy of polycosanol with aspirin an attractive option. (Arruzazabala et al. (1997) Pharmacol. Res. 36293-297). For other reports on the anti-thrombic effects of polycosanol compositions see Arruzazabala et al. (2002) Clin. Exp. Pharmacol. 29:891-7; Janikula (2002) Alternative Medicine Review 7:203-217; and Stusser et al. (1998) Int J Clin Pharmacol Ther 36(9):469-73). For reports on other cardiovascular benefits of polycosanol compositions see Noa et al. (1997) J. Pharm. Pharmacol. 49:999-1002; Noa et al. (2001) Pharmacolo. Res. 43:31-37; Molina et al. Braz. J. Med. Biol. Res. 32:1269-1276; Janikula (2002) Alternative Medicine Review 7:203-217; and Menendez et al. (2002) Can J Physiol Pharmacol. 80:13-21.
Polycosanol(s) and polycosanolic acids have also been reported to be effective as nutritional and therapeutic preparations for the prevention and treatment of aging and related conditions, such as, atherosclerosis, hypertension, diabetes, tumors, obesity, overweight, hypertriglyceridemia, hypercholesterolemia, as well as other conditions. (Pistolesi, WO 02/052955 (2001)). There are a numerous other reported uses of individual polycosanols and mixtures thereof in the literature. This provides a significant incentive to develop new sources containing novel polycosanol compositions of matter, which would be expected to have different pharmacological effects and strengths. The multitude of uses for the individual alcohols and mixtures thereof, also provides a significant incentive to develop improved methods for isolating these compounds.
Polycosanol has been determined to be safe at a dosage of up to 500 mg/kg/day, which is 1500 times greater than the standard human dosage of 20 mg/day. Rats treated with a dosage of 500 mg/kg/day for 12 to 24 months exhibited no signs of toxicity or carcinogenesis resulting from treatment with polycosanols. (Aleman et al. (1995) Food Chem. Toxicol. 33:573-578). Dogs given 180 mg/kg/day for one year showed no side effects resulting from the composition (Mesa et al. (1994) Toxicol. Lett. 73:81-90) and monkeys given 25 mg/kg/day for 54 months showed no signs of adverse effects (Rodrigurz et aL (1994) Food Che. Toxicol. 32:565-575). In reproductive and fertility studies, polycosanol compositions exhibited no adverse effects on fertility, reproduction and development in rats fed up to 500 mg/kg/day for two weeks before mating, throughout pregnancy, and 21 days into lactation, and in male rats given 500 mg/kg/day for 60 days prior mating (Rodriguez and Garcia (1998) Teratog. Carcinog. Mutagen. 18:1-7). Rabbits treated with a dosage of 1000 mg/kg/day during pregnancy showed no evidence of teratogenic and embryonic toxicity. The tissue distribution of polycosanol in animal models has been reported by Kabir and Kimura. ((1995) Ann. Nutr. Metab. 39:279-284 and (1993) 37:33-38). Polycosanol has been shown to be stable in 10 mg tablets for up to nine months (Cabrera et al. (2002) Boll. Chim. Farm. 141:223-229) with no interaction with excipients (Cabrera et al. (2002) Boll. Chim. Farm. 141:138-142).
Cholestin(trademark), a dietary supplement from Pharmanex, contains octacosanol isolated from the wax of honey bees. This product has been shown to promote healthy cholesterol levels by inhibiting the production of cholesterol in the liver. LesstanoL(trademark) brand from Garuda International, Inc. contains natural octacosanol (95%) isolated from sugar cane or vegetable waxes. TwinLab Octacosanol Plus is derived from spinach, a superior and all natural source of octacosanol. Octacosanol in Nature""s Way""s products is a naturally occurring substance found in sugar cane, wheat germ oil, spinach, and other natural sources. Octacosanol from Viable Herbal Solutions is the active ingredient in wheat germ oil and is used to increase endurance, stamina and vigor. Applicant is not aware of any reports regarding the production polycosanol compositions from Ericerus pela wax.
Sierra et al. has developed a gas chromatographic (GC) method for determining the fatty alcohol content of film-coated tablets. (Sierra et al. (2002) J. AOAC Int. 85:563-566). 1-Octacosanol in rat plasma has been quantified after solid phase extraction and derivatization using a capillary GC method developed by Marrero and Gonzalez ((2001) J. Chromatogr. B Biomed. Sci. Appl. 762:43-49). The polycosanols were derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide. The general physical characteristics of sugar cane polycosanols have been reported by Uribarri et al. ((2002) Drug Dev. Ind. Pharm. 28:89-93).
It is an objective of this invention to provide a mixture of higher primary aliphatic alcohols, referred to herein as xe2x80x9cpolycosanol(s)xe2x80x9d having a unique chemical composition profile.
It is another objective of this invention to provide an improved method for obtaining a highly pure mixture of higher primary aliphatic alcohols.
The present invention includes a novel method for the preparation of a unique profile of primary aliphatic alcohols, having 24 to 30 carbon atoms, from the wax secreted by the insect Ericerus pela . Included in the present invention is the composition of matter, referred to herein as xe2x80x9cpolycosanolxe2x80x9d produced by the method of this invention. The polycosanol composition produced by the method of this invention is comprised primarily of two major components, the primary aliphatic alcohols, hexacosanol and octacosanol, with two minor components, the primary aliphatic alcohols, tetracosanol and triacontanol. Further included in this invention is the use of said composition of matter for the prevention and treatment of obesity, syndrome X, diabetes, hypercholesterolemia, atherosclerotic complications, ischemia and thrombosis.
The method of the present invention is comprised of the steps of (a) hydrolyzing the melted wax obtained from the insect with a base; (b) neutralizing the basic hydrosylate obtained from step (a) to yield a lower purity composition of matter comprised of polycosanols; and (c) optionally extracting the hydrosylate without neutralization with an organic solvent to obtain a higher purity polycosanol composition. In another embodiment of the instant invention, the method further comprises the step of (d) purifying the hydrolyzed product obtained in step (c) by recrystallization. The method of this invention can be extended to the isolation and purification of polycosanol compositions from any source of wax, in particular from any source of insect wax.
The present invention includes the novel mixtures of primary aliphatic alcohols (referred to herein as xe2x80x9cpolycosanolxe2x80x9d) prepared and isolated by the methods of this invention. In one embodiment, the composition of matter is isolated following hydrolysis of the wax and neutralization with no further purification. This composition is comprised of approximately 35-55% of the long chain primary aliphatic alcohols of interest. The major components of this composition are: 1-hexacosanol (xcx9c20-30%) and 1-octacosanol (xcx9c15-25%) and the minor components are: 1-triacontanol (xcx9c2-4%) and 1-tetracosanol (xcx9c1-3%). In another embodiment, the composition of matter is isolated following hydrolysis of the wax and further purification via extraction of the polycosanols under basic conditions with an organic solvent. In this embodiment the composition of matter is comprised of approximately 75-100% of the long chain primary aliphatic alcohols. The major components of this composition are 1-hexacosanol (xcx9c30-50%), 1-octacosanol (xcx9c25-45%), 1-triacontanol (xcx9c4-10%) and 1-tetracosanol (xcx9c3-9%). In yet another embodiment, the composition can be even further purified by recrystallization. As noted above, the alcoholic mixtures obtained from Ericerus pela wax in accordance with the present invention can be distinguished from all other currently known sources of polycosanols. Table 1, below highlights the differences between the compositions isolated from sugar cane and bee wax, which are currently the major commercially available sources of polycosanols, with the composition isolated according to the method of this invention.
The present invention also includes the use of the polycosanol compositions isolated by the method of this invention for the prevention and treatment of obesity, syndrome X, diabetes, hypercholesterolemia, atherosclerotic complications, ischemia and thrombosis. These novel compositions are expected to have different pharmacological effects and strengths. The compositions can be formulated in a pharmaceutical composition, foodstuff or dietary supplement and administered to humans and other animals.
The present invention provides not only a novel source of polycosanols and the resulting novel compositions of matter, but also provides an improved method for extracting these compounds from a wax. Preferred levels of certain operational parameters in the extraction and purification process have been discovered which lead to further enhancement of the purity level of the isolated alcohols and enhancement of the percent recovery of the alcohols from Ericerus pela wax. These operational parameters include optimized solvent volume and quantity of basic solution in the hydrolysis process; and extraction of the basic powder of crude polycosanol with lower polarity organic solvents than those used in current methods, which is critical to obtaining higher quality insect polycosanol.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.